Abstract An important limitation of topical 5-aminolevulinic acid (ALA)-based photodetection and photodynamic therapy is that the amount of the fluorescing and photosensitizing product protoporphyrin IX (PpIX) formed is limited. The reason for this is probably the limited diffusion of ALA through the stratum corneum. A solution to this problem might be found in the use of ALA derivatives, as these compounds are more lipophilic and therefore might have better penetration properties than ALA itself. Previous studies have shown that ALA hexyl ester (ALAHE) is more successful than ALA for photodetection of early (pre)malignant lesions in the bladder. However, ALA pentyl ester slightly increased the in vivo PpIX fluorescence in early (pre)malignant lesions in hairless mouse skin compared to ALA. The increased PpIX fluorescence is located in the stratum corneum and not in the dysplastic epidermal layer. In the present study, ALA- and ALAHE-induced PpIX fluorescence kinetics are compared in the normal nude mouse skin, of which the permeability properties differ from the bladder. Application times and ALA(HE) concentrations were varied, the effect of a penetration enhancer and the effect of tape stripping the skin before or after application were investigated. Only during application for 24 h, did ALAHE induce slightly more PpIX fluorescence than ALA. After application times ranging from 1 to 60 min, ALA-induced PpIX fluorescence was higher than ALAHE-induced PpIX fluorescence. ALA also induced higher PpIX production than ALAHE after 10 min of application with concentrations ranging from 0.5 to 40%. The results of experiments with the penetration enhancer and tape stripping indicated that the stratum corneum acts a barrier against ALA and ALAHE. Use of penetration enhancer or tape stripping enhanced the PpIX production more in the case of ALAHE application than in the case of ALA application. This, together with the results from the different application times and concentrations indicates that ALAHE diffuses more slowly across the stratum corneum than ALA.